Opiate abuse is an epidemic in the US, where opiates have a high relapse rate, and unintentional overdose deaths have tripled from 2001 to 2013. Despite the prevalence of opiate abuse, relatively little is known about the neuroadaptations that occur with chronic use. We previously determined that chronic opiate exposure induces a unique change in the size of dopamine (DA) neurons in the ventral tegmental area (VTA), a key brain region in the mesocorticolimbic reward circuit, and decreased VTA DA soma size was also observed in post- mortem human samples of heroin addicts, suggesting translational relevance. Moreover, we have demonstrated that changes in soma size are correlated with VTA DA neuronal activity and reward processing in rodent models, suggesting a functional link. However, further understanding of opiate-induced structural and functional neuroadaptations of VTA DA neurons has been limited by VTA cellular heterogeneity and techniques to isolate and examine specific subsets of VTA DA neurons. For example, unbiased genomic approaches necessary to identify novel molecular mediators have been limited to homogenization of the entire VTA, which includes multiple neuron types, not just DA cells. Further, it has become increasingly clear through optogenetic studies that VTA DA neurons themselves are diverse, as subsets of VTA DA neurons are differentially activated by rewarding or aversive stimuli depending on their projection target, e.g. the nucleus accumbens (NAc) or prefrontal cortex (PFC). Thus, this proposal seeks to address the limitations stemming from VTA cellular heterogeneity through the use of cutting-edge viral and molecular techniques, addressing the central hypothesis that chronic morphine induces structural plasticity in VTA DA neurons in a projection-specific manner and is mediated by transcriptional changes in VTA DA neurons. Aim 1 will determine whether chronic morphine induces circuit-specific changes in structural plasticity (soma size and dendritic spine density) through injection of retrograde adeno-associated vectors that express fluorescent proteins in a Cre-recombinase dependent manner into the NAc and PFC of tyrosine hydroxylase (TH)-Cre mice. Preliminary data suggest differences in basal and morphine-induced soma size between NAc- and PFC-projecting VTA DA neurons. Aim 2 will use an innovative cell type-specific purification technique, Translating Ribosome Affinity Purification (TRAP) to isolate mRNA specifically from VTA DA neurons and assess gene expression changes in candidate genes, as well as novel genes via RNA-sequencing, that may mediate morphine-induced structural and functional adaptations. Our previous RNA-sequencing data have shown that chronic morphine induces a unique gene expression pattern in the VTA compared to cocaine; these studies will determine whether such changes are driven specifically by VTA DA neurons. Together, this work is expected to advance the understanding of how chronic opiate exposure alters the structure and function of VTA DA neurons and to identify novel mechanisms underlying mesocorticolimbic circuit dysfunction.